Project Summary Plasmodium falciparum is a deadly human parasite that causes malaria and is responsible for nearly 450,000 deaths every year. Malaria is endemic in large regions of the world, home to about 4 billion people and it affects ~250 million people annually. There are no effective vaccines against malaria and antimalarial drugs are the mainstay of treatment. At this time, the parasite has gained resistance to all clinically available antimalarial drugs and these drug-resistant strains are spreading throughout the world, threatening all the progress that has been made against this disease in the last decade. Therefore, it is imperative that we constantly generate new drugs and identify potential drug targets to stay ahead of this nefarious disease. The clinical manifestations of this devastating parasitic disease, including death, are caused by the growth of P. falciparum within the host red blood cell (RBC). To build a suitable habitat for growth inside RBCs, the malaria parasite completely transforms the host cell. It changes the metabolism of the RBC, makes the RBC more rigid such that it is harder for the infected RBC to pass through capillaries, modifies the RBC membrane to allow for favorable movement of nutrients, and alters the binding properties of the RBC so that the infected cell can bind to the endothelial cells lining blood vessels. The sum of these changes leads to disease and death, for instance, binding of the P. falciparum infected RBC to endothelial cells can clog blood vessels in the brain leading to clots that eventually result in death. The subjugation of the infected RBC is accomplished through the action of several hundred proteins that the parasite transports to the host cell via poorly understood mechanisms. The export of parasite effector proteins is essential for transforming the RBC and therefore, for causing disease. Parasite effector proteins that are synthesized in the parasite cytoplasm need to be transported across three or four cellular membranes in order to reach their site of action in the host RBC. The molecular mechanisms that recognize, sort, and transport these parasite effectors to the infected RBC remain to be identified. The proposed studies aim to unravel the molecular processes that govern key early events that set parasite effectors on the path to the host RBC. We will pursue two aims to accomplish this goal. First, we will generate conditional mutants of proteins in the endoplasmic reticulum of the parasite that are potentially required for export of parasite effectors. The mutants will be analyzed using genetic, cellular, and biochemical approaches to determine their roles in the export of parasite proteins. Second, we will take an unbiased interactome screening approach that uses a proximity-based labeling approach and discover proteins that usher exported proteins to their site of action in the host RBC. Attaining the objectives of the research program will reveal key and unique protein trafficking mechanisms of P. falciparum that may be targeted for antimalarial drug development.